The macula is located at the back of the eye in the center of the retina. When the millions of cells in this light-sensitive, multilayer tissue deteriorate, central vision is lost along with the ability to perform tasks such as reading, writing, driving, and seeing color. This “macular degeneration” principally affects the elderly and has a prevalence of about 3% in populations between 75-79 years of age and about 12% for populations over 80 years of age.(1) In younger populations, macular degeneration is found in individuals with genetic disorders, such as Stargardt, Vitelliform or Best (VMD), Sorsby's Fundus Dystrophy and Malattia Leventinese (Doyne Honeycomb or Dominant Radial Drusen). Stargardt Disease is the most common form of inherited juvenile macular degeneration, affecting about 1 in 10,000 children.
There are no therapies currently available for genetic or dry (non-neovascular) age related macular degeneration. Vitamin supplements such as antioxidants, and diet changes, such as low fat diets, have been shown to slow disease progression in some clinical studies. However, for the majority of patients, diagnosis is followed by the progressive loss of central vision. (2-4)
The above listed macular dystrophies are all marked by the accumulation of lipofuscin, fluorescent deposits, in the retinal pigment epithelium (RPE) cell layer (5-11). The only compounds that have been characterized to date from RPE lipofuscin, A2E (N-retinylidene-N-retinylethanolamine) and ATR-dimer (all-trans-retinal dimer), are derived from reactions of all-trans-retinal, an isomer of 11-cis-retinal, the chromophore of the visual pigments (12, 13). These toxic retinal dimers have been shown to cause RPE cell death, which is thought to lead to photoreceptor cell degeneration and vision loss (2, 14-22). Slowing the visual cycle using RPE 65 antagonists (23-25) or retarding the delivery of vitamin A (retinol) to the RPE by, e.g., limiting dietary intake of vitamin A (26) or blocking serum retinol binding proteins (27) have been shown to impede or abolish RPE lipofuscin formation in animal models. Conversely, increasing the amount of all-trans-retinal in outer segments, such as occurs with the mutations in abcr−/− (responsible for recessive Stargardt disease), leads to the rapid accumulation of lipofuscin pigments (8).
The above cited evidence leads to the conclusion that lipofuscin and/or A2E and ATR-dimer in RPE cells may reach levels that contribute to a decline in cell function followed by vision loss and that vitamin A plays an important role in ocular lipofuscin formation.(28)
Several vitamin A analogs have been shown to limit lipofuscin formation in a mouse model by slowing down the visual cycle. The ABCR−/− mouse model has been used to test approaches to limit RPE lipofuscin formation. Mice lacking the ABCR (also known as ABCA4) gene encoding a photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter, lack the ability to properly shuttle vitamin A (in the form of retinal) in the eye. ABCR−/− mice were developed as an animal model of human recessive Stargardt's Disease (8). As in humans with recessive Stargardt's disease, ABCR−/− mice accumulate large lipofuscin deposits in RPE cells of their eyes, as shown in FIG. 1, and eventually experience delayed dark adaptation. Lipofuscin accumulation and vision loss observed in this mouse model is also thought to be relevant to age-related macular degeneration (AMD) and other macular dystrophies.
In attempts to slow down the visual cycle, visual cycle enzymes have been targeted. In such approaches, small molecules have been proposed as antagonists to visual cycle enzymes. In this methodology, the drug (often a vitamin A derivative) binds to visual cycle proteins, which blocks participation by the proteins in the visual cycle, and slows the visual cycle. Another approach to slowing the visual cycle includes impeding the delivery of vitamin A from the blood to the eye.
Drug candidates for inhibiting lipofuscin formation by slowing down the visual cycle have been evaluated by their ability to cause delayed dark adaptation (a side effect of an impaired visual cycle) and their ability to impede the age related accumulation of eye lipofuscin as measured by the concentration of A2E and other byproducts of the visual cycle, e.g., ATR-dimer. Examples of such drug candidates and their corresponding mechanisms of action to slow the visual cycle are summarized below:
13 cis-retinoic acid (Acutane or isotretinoin) is thought to inhibit the enzymes 11-cis-retinol dehydrogenase and RPE65 involved in the visual cycle.(19, 24) When administered, 13 cis-retinoic acid has been shown to cause delayed dark adaptation. When 3-month-old ABCR knockout mice (n=3) were administered 13-cis-retinoic acid at 40 mg/kg/day for one month, the mice showed a decrease in A2E formation by about 40-50% compared to control ABCR knockout mice (n=3).(24) Furthermore, when 2-month-old ABCR knockout mice were administered 13-cis-retinoic acid at 20 mg/kg/day for two months, the mice also showed a decrease in lipofuscin formation in the RPE cell layer as judged by electron microscopy.
(12E,16E)-13,17,21-trimethyldocosa-12,16,20-trien-11-one (TDT) is thought to inhibit RPE65. (23) When TDT is administered to mice, delayed dark adaptation is observed. When 2-month-old ABCR knockout mice (n=2) were administered TDT at 50 mg/kg bi-weekly for two months, they showed a decrease in A2E formation by about 50-85% compared to control ABCR knockout mice (n=2).(23)
(2E,6E)-N-hexadecyl-3,7,11-trimethyldodeca-2,6,10-trienamine (TDH) is also thought to inhibit RPE65 and when given to mice, causes delayed dark adaptation.(23) When 2-month-old ABCR knockout mice (n=2) were administered TDH at 50 mg/kg bi-weekly for two months, they showed a decrease in A2E formation by about 50% compared to control ABCR knockout mice (n=2).(23)
All-trans-retinylamine (Ret-NH2) is thought to inhibit RPE65 and when administered to mice results in severe delayed dark adaptation. When 1-month-old ABCA4 knockout mice were given Ret-NH2 at 40 mg/kg bi-weekly for two months, they showed a decrease in A2E formation by about 50% compared to control ABCA4 knockout mice.(25)
N-(4-hydroxyphenyl)retinamide (Fenretinide) slows the influx of retinol into the eyes by reducing levels of vitamin A bound to serum retinol-binding protein. Thus, treatment with Fenretinide lowers the levels of vitamin A in the eye which leads to delayed dark adaptation. When 2-month-old ABCA4 knockout mice (n=3) were administered Fenretinide at 20 mg/kg/day for one month, they showed a decrease in A2E formation by about 40-50% compared to control ABCA4 knockout mice (n=3).(27)
Thus, recently proposed therapeutic approaches to limit lipofuscin formation are based upon slowing the visual cycle (1, 10, 11, 12). There are, however, disadvantages to slowing the visual cycle as a means to impede lipofuscin formation in order to prevent, e.g., macular degeneration. Four of these disadvantages are detailed below.
One immediate disadvantage of slowing the visual cycle is that it leads to delayed dark adaptation and poor vision in dim light or at night. Poor night vision (Scotopic dysfunction) is already a functional marker of early age-related maculopathy (ARM), and has been linked to the occurrence of falls and vehicle collisions.(29, 30) A further slowing down of the visual cycle is expected to make night vision worse in patients who already suffer from poor night vision.
Second, in order to sufficiently impede lipofuscin formation, one would have to slow down the visual cycle for a prolonged period of time. But, long-term slowing of the visual cycle leads to photoreceptor cell death and loss of vision.(31, 32) In fact, impairment of the visual cycle is the cause of various retinal diseases such as Stargardt's disease, retinitis pigmentosa, Lebers Congenital Amaurosis, Fundus Aibipunctatus, age-related macular degeneration and Congenital Stationary Night Blindness.
Third, the above methodology often uses vitamin A analogs to impede proteins involved in vitamin A processing in the eye. However, vitamin A analogs of diverse structures, pharmacological profiles, receptor affinities, and biologic activities have been shown to be toxic. Indeed, numerous vitamin A analogs have been shown in experimental animal models, cellular models, epidemiological data and clinical trials to inhibit or retard various biological functions such as, for example, bone growth, reproduction, cell division, cell differentiation and regulation of the immune system.(33-37). Thus, inhibiting vitamin A processing in the eye is also expected to retard some of these basic bodily functions, which may lead to significant adverse side effects.
For example, vitamin A analogs that are currently used to treat certain cancers and/or psoriasis such as, e.g., Bexarotene (Targretin), Etretinate (Tegison) Acitretin (Soriatane), Fenretinide (N-(4-hydroxyphenyl) retinamide or 4-HPR) and 13 cis-retinoic acid (Accutane or isotretinoin) have side effects, which include, e.g., dry nose, nosebleeds, chapped lips, mouth sores, increased thirst, sore tongue, bleeding gums, dry mouth, cold sores, dry or irritated eyes, dry skin, peeling or scaly skin, hair loss, easy bruising, muscle aches, nausea, stomach upset, cough or swelling of the hands or feet, vision problems, chest pain, tightness in the chest, abnormal pulse, dizziness, vomiting, severe headache, and yellowing of the eyes/skin (jaundice).(38-40)
Fourth, in animal models, candidate dugs for limiting lipofuscin formation have been shown to be effective only in relatively large doses. For example, studies in mouse models typically use doses around 11-40 mg/kg/day. But, current vitamin A analogs are typically used in the clinic at doses of 1-3 mg/kg/day to minimize side effects. Larger dosing regimes will lead to increased side effects.
For example, Fenretinide (N-(4-hydroxyphenyl)retinamide or 4-HPR) is currently used to treat cancer and clinical trails are in progress for AMD. The most common adverse effects reported among 1,432 patients who underwent treatment with this drug at a dosage of 200 mg/day for a five year period were: diminished dark adaptation, cumulative incidence (16%) and dermatologic disorders (16%). Less common effects were gastrointestinal symptoms (8%) and disorders of the ocular surface (8%).(41) At this relatively low dose a delay in dark adaptation, an assessment of the effectiveness of a drug to impede lipofuscin formation, was only observed in 16% of patients. In another study when patients were administered larger oral doses of 600 or 900 mg/m2 bid in 6-week cycles, mild to moderate adverse effects were reported in 43 (95%) of the 45 patients that were possibly linked to Fenretinide. These side effects included: fatigue, headache, skin changes (dry skin, pruritus, and rash) and digestive tract symptoms (abdominal pain, cramping, diarrhea, stomatitis, and xerostomia). Grade 2 toxicities reported as possibly linked to Fenretinide treatment included seizures and confusion.(42)
A patient with an anaplastic astrocytoma who had been receiving treatment with Fenretinide at the 600 mg/m2 bid dose for one cycle presented with headaches, nausea, and vomiting, and was found to have a small intracranial bleed in the region of the basal ganglia. He recovered without deficits and continued treatment without further events. Another patient, who was also undergoing treatment at the 600 mg/m2 bid dosage and was receiving oral anticoagulation with warfarin for deep venous thrombosis, died after developing an uncontrollable nasal bleed (international normalized ratio >6.0). Of the four patients treated at the 900 mg/m2 bid dose, one had grade 3 vomiting, grade 2 speech impairment, and grade 1 memory impairment, which improved without residual symptoms.(42)
A typical dose of 13 cis-retinoic acid (Accutane or isotretinoin) for treatment of acne is 0.5 to 1 mg/kg/day for children and 2.0 mg/kg/day for adults for fourteen days in a row, followed by a 14-day break. This twenty-eight day course is usually repeated five more times.(40) These doses are about 10 to 80 times less than what was used in mice to impede A2E formation.(24) For larger doses of 13 cis-retinoic acid used to treat cancer, side effects occurring in more than 30% of users included headache, fever, dry skin, dry mucous membranes (mouth, nose), bone pain, nausea and vomiting, rash, mouth sores, itching, sweating, and eyesight changes.(43, 44)
Side effects occurring in 10-29% of users of 13 cis-retinoic acid include back pain, muscle and joint pain, allergic reaction, abdominal pain, poor appetite, dizziness, drowsiness, insomnia, anxiety, numbness and tingling of hands and feet, weakness, depression, hair loss (thinning), dry eyes, sensitivity to light (see eye problems), decreased night vision, which may persist after treatment is stopped, feet or ankle swelling, and low blood counts.(44) In addition, it has been observed that white and red blood cells and platelets may temporarily decrease, which may put a patient at increased risk for infection, anemia and/or bleeding. Side effects also include abnormal blood tests: increased triglyceride, cholesterol and/or blood sugar levels.
The above disadvantages make slowing of the visual cycle a difficult methodology to adapt alone for the clinical treatment of ophthalmologic disorders, e.g., macular degeneration.